The major objective of this proposal is to develop an oral live E. coli vaccine, using recombinant DNA techniques, for the control of enterotoxigenic E. coli (ETEC)-mediated diarrheal disease. A natural human ETEC strain will first be deprived of virulence plasmids mediating production of heat-labile toxin (LT), heat-stable toxin (ST) and pilus colonization factors (PCF): CFA I or CFA II. We will then prepare a vaccine from this plasmid-deprived strain by re-inserting plasmid vectors carrying cloned DNA sequences of appropriate antigens. We have developed an adult rabbit model of acute, fatal, dehydrating diarrhea due to ETEC or Vibrio cholerae infection: the Removable Intestinal-Tie Adult Rabbit Diarrhea (RITARD) model. This model will be used to evaluate virulence factors of ETEC and the effectiveness of vaccines against infection with these organisms. The specific objectives of this project are as follows: 1) Using natural human LT/ST ETEC strains, we will characterize the dose-response in RITARD model animals, the peak immune responses to LT, PCF and O antigens, the time course of the immune response and the protection it engenders, and the kinetics of colonization during the first 24 hours after challenge. We will 2) induce the loss of LT and/or ST in these strains and observe the resulting changes in colonization, infection kinetics, immune response and protection. 3) Prepare cloned plasmids containing the genes for defective LT or LT-subunits and either CFA I or CFA II; or both, and identify a natural ETEC strain to serve as their host. 4) Characterize the stability of the plasmid(s) in the host strain and the extent to which the defective LT and PCF are expressed in vitro. 5) Characterize colonization, immune response and protection against ETEC challenges following infection by the vaccine strain(s).